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At a press event promoting the release of "Ant-Man" in China, Marvel representatives told the audience it is developing Chinese characters to add to its roster of heroes and villains.
According to a report by the Xinhua News Agency (translated by GB TImes),Marvel's Manager of Licensed Publishing Jeffrey Reingold and VP of Animation Steve Wacker told the crowd that in addition to bringing its already-existing catalog of characters to China, Marvel is working on adding new, Chinese characters to its universe.
CBR News has reached out to Marvel for further information.
"Ant-Man" Is Bigger Than "Iron Man," "Captain America" or "Thor" in China
Marvel continues to grow in popularity in China, where "Ant-Man" opened in the number one position for the weekend, bringing in $43.5 million to make it the second-biggest Marvel/Disney opening in that country, after "Avengers: Age of Ultron."
"We have felt the passion of Chinese fans," Reingold said. "As long as there is a demand in the market, it is possible to add Chinese elements and culture to Marvel products and create Chinese heroes and stories."
Discuss this story in CBR's Marvel Comics forum. | 95 Comments


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Scullers on the Potomac River.
Credit: Brian G. Green
Creative #: ngs0_4135
Available for Editorial uses. Editorial use: To report or illustrate a newsworthy event or matter of general interest. Editorial uses do not typically require a model or property release.Learn more
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Scullers On The Potomac River Stock PhotoAdults Only,Blurred Motion,Color Image,Day,Directly Above,Four People,Horizontal,Outdoors,Panning,Photography,Potomac River,Rowboat,Rowing,Scull,Sports Clothing,Sports Race,Sweep Rowing,Transportation,Uniform,Washington DCPhotographer Brian G. GreenCollection: National Geographic

subset: commoncrawl


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At LECOM Medical Fitness & Wellness Center, we believe a peaceful mind and a calm spirit are just as important as a healthy body. In keeping with the Osteopathic philosophy of nurturing the mind, body and spirit, we approach health and fitness from a holistic, medically integrated perspective. We offer something for every wellness goal, from day-to-day health and fitness, to sports performance training and rehabilitation. These services include:
- FREE Fitness Assessment with a certified fitness specialist
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- Certified Medical Fitness Facility
Our facility will offer you a unique and personalized experience not offered anywhere else. Come in today and experience the LECOM Medical Fitness & Wellness Center.Learn More
As places committed to healing and lifelong well-being, the LECOM Medical Fitness & Wellness Center and the Lake Erie College of Osteopathic Medicine will be accepting submissions for our 5th Annual "Art of Healing" art show. All artwork will be on public display from August 20th – 26th at the LECOM Medical Fitness & Wellness Center. If you […]full story
The LECOM 5K will be held Saturday, August 6th at 9:oo am. You can pre-register now. All registrations received by August 1st will include a race t-shirt. You can sign up online here . Or print and complete the registration form and return it along with payment to LECOM Medical Fitness & Wellness Center.full story
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text: Photos: Lauren Bacall's Life on Screen and Stage Aug. 12, 2014 10:36 p.m. ET 0 COMMENTS fullscreen Lauren Bacall made her film debut in 1944's 'To Have and Have Not,' starring her future husband, Humphrey Bogart. Everett Collection … Bacall and Bogart in a scene from 'To Have and Have Not.' Ronald Grant Archive/Everett Collection … Vice President Harry S Truman played the piano during Bacall's appearance at the National Press Club canteen in Washington in February 1945. Associated Press … Bacall and director Herman Shumlin in a between-scenes huddle during the filming of 'Confidential Agent' in July 1945, joined by Bogart. Associated Press … Bacall starred in 1957's 'Designing Woman.' Everett Collection … Bacall and actor Walter Matthau held the Tony award she won for best actress in a musical comedy in April 1970 for the Broadway hit 'Applause.' Ron Frehm/Associated Press … Bacall attended the 1977 RFK celebrity tennis tournament at Forest Hills, N.Y. Associated Press … Bacall joined Henry Kissinger, left, and actor Henry Fonda at a party to launch her autobiography, 'By Myself,' at the Palace Theater in New York in June 1979. Ron Frehm/Associated Press … Bacall rehearsed with Eivind Harum for the musical 'Woman of the Year' in New York in January 1981. Suzanne Vlamis/Associated Press … Bacall, alongside actor Anthony Hopkins, held the Cecil B. DeMille Award for Lifetime Achievement that she won at the 50th annual Golden Globe Awards in Beverly Hills, Calif., in January 1993. Reed Saxon/Associated Press … Bacall with her children Leslie and Stephen at a ceremony honoring her late husband, Humphrey Bogart, with an honorary postage stamp at Mann's Chinese Theatre in Hollywood, Calif., in July 1997. Fred Prouser/Reuters … Bacall with her dog Sophie during a photo call to present her film 'The Walker' at the 57th Berlinale International Film Festival in Berlin in February 2007. Arnd Wiegmann/Reuters … Bacall; cinematographer Gordon Willis, right; and filmmaker Roger Corman with their Honorary Oscars at the Academy of Motion Picture Arts & Sciences Governors Awards in Hollywood, Calif., in November 2009. Fred Prouser/Reuters … Replay Photos: Lauren Bacall's Life on Screen and Stage More Slideshows Photos: Deadly Hot-Air Balloon Crash in Texas Photos of the Day: July 29 of SHOW CAPTION HIDE CAPTION fullscreen Show More Archives


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Chili Mac RecipeRead Reviews Be the first to add a review
- 1 pound lean ground beef (90% lean), cooked and drained
- 2 cans (16 ounces each) hot chili beans, undrained
- 2 large green peppers, chopped
- 1 large onion, chopped
- 4 celery ribs, chopped
- 1 can (8 ounces) no-salt-added tomato sauce
- 2 tablespoons chili seasoning mix
- 2 garlic cloves, minced
- 1 package (7 ounces) elbow macaroni, cooked and drained
- Salt and pepper to taste
- In a 5-qt. slow cooker, combine the first eight ingredients. Cover and cook on low for 6 hours or until heated through. Stir in macaroni. Season with salt and pepper. Yield: 6 servings.
Reviews for Chili Mac
"my husband and I did not care for it-very disappointing"
"Ok, we didn't care for it much."
"I realy loved this one, it had just the right amount of heat, and I love spicey food"
"This was so easy to put together and I loved how it cooks in the slow cooker. I ate mine with a big of hot sauce dashed in it."
"My family loves this easy supper. We dont like it too hot so I put red kidney beans in it. Also, I put diced tomatoes with chilies in it. I use wheat noodles instead of white to make it a little healthier. Serve with cheddar cheese, sour cream and maybe a sandwich! Great way to warm up on a cold day!"
"Definitely a winner in my house! I substituted kidney beans for the hot chili beans since I have small children, but it was really good! A little cheese on top and it's a meal!"
"A nice recipe; I love a good chili mac. I'll definitely make again, although I didn't use the crockpot."
"simple and yummy."
"was very good and simple!"
"My family loved this recipe and said I should make it often. I didn't cook it in the crockpot because I didn't get it in there in time. It turned out great."


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HULL -- The Sergeant Bobby Guiney Memorial Motorcycle Run will begin on Saturday, May 17 at 11 a.m. at the Red Parrot Restaurant in Hull. Registration for the ride in memory of Boston Harbor Master Sgt. Bobby Guiney will be held at 8:30 a.m. at the Red Parrot the day of the ride. Costs are $25 for riders and $15 for passengers. Complimentary t-shirts will be provided to the first 300 registered riders and food will be provided at the Red Parrot following the ride.
If you wish to ride and do not have a motorcycle, or if you wish to take another rider email email@example.com. For details go to bostonbiker.com.
Note: First and last names are required for commenting. If you do not provide your first and last name when signing up, you will be banned.
Location, ST | website.com


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The significant amount of energy stored in a large high-field superconducting magnet can be sufficient to destroy the coil in the event of an unprotected quench. For magnets based on high-temperature superconductors (HTSs), such as Bi2Sr2CaCu2Ox (Bi2212) and YBa2Cu3O7-x (YBCO), quench protection is particularly challenging due to slow normal zone propagation. A previous computational study showed that the quench behavior of HTS magnets is significantly improved if the turn-to-turn electrical insulation is thermally conducting, enhancing 3-D normal zone propagation. Here, a new doped-titania electrical insulation with high thermal conductivity is evaluated. The thermal conductivity of the insulation is measured at cryogenic temperatures, and its chemical compatibility with Bi2212 round wires is determined. Thin layers of the insulation are deposited onto the surface of Bi2212 and YBCO wires, which are then wound into small coils to study the quench behavior. Results show that the critical current and homogeneity of Bi2212 coils are improved relative to coils reacted with mullite insulation. Relative to similar coils with conventional insulation (mullite for Bi2212 and Kapton for YBCO), the turn-to-turn quench propagation is increased by a factor of 2.8 in Bi2212 coils at 4.2 K and self-field and by a factor of 2.5 in YBCO coils at 4.2 K and 5 T. These results indicate that doped-titania insulation may significantly improve Bi2212 and YBCO coils. Increased normal zone propagation velocity enhances quench detection and quench protection, and the thinness of the insulation relative to the most common alternatives increases the magnet winding pack current density and reduces the coil specific heat.
A not-for-profit organization, IEEE is the world's largest technical professional organization dedicated to advancing technology for the benefit of humanity.© Copyright 2016 IEEE - All rights reserved. Use of this web site signifies your agreement to the terms and conditions.


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Warning: file_get_contents(http://maps.google.com/maps/geo?&q=new%20york&output=csv&key=ABQIAAAAO8mFjDAFjx7wE9qxCv_VfxSBGrErVhUFOrvl_oHMAC0O0MFsVxQ2iPALXtw3LYf7zBGbxk9vedGuNA): failed to open stream: HTTP request failed! HTTP/1.0 404 Not Found in /home/web/forum.photojojo.com/googleMapClass.php on line 1343xstevex (a.k.a. "steve")
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Stock photography and/or related sites?
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yea Alicia , I have a friend who does it full time and shes done some high profile musicians , CD...
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dough ?....doh!!
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Israel banned from showing holiest site on tourism adverts
The Advertising Standards Agency said that a picture used of Jerusalem was misleading becuase the territory is disputed.
Israel has been banned from showing images of the Western Wall in tourism adverts, after a ruling by the Advertising Standards Agency.
An image on the advert for holidays in Israel showed a picture of the Kotel, with the gold Dome of the Rock in the background, captioned "Jerusalem"'.
The ASA received a complaint that the picture showed disputed east Jerusalem, and misleadingly portrayed it as part of Israel.
In its adjudication, the ASA ruled that the advert breached its truthfulness guidelines and said the advert must not be used again.
It said: "The ASA noted the image of Jerusalem used in the ad featured the Western Wall of the Temple Mount and the Dome of the Rock, which were both in East Jerusalem, a part of the Occupied Territories of the West Bank.
"We understood, however, that the status of the occupied territory of the West Bank was the subject of much international dispute, and, because we considered that the ad implied that the part of East Jerusalem featured in the image was part of the state of Israel, we concluded that the ad was likely to mislead."
A spokesman for Israel's Ministry of Tourism said that it was "entirely accurate to assert that a visitor to Israel could visit Jerusalem as part of a short visit."
The ministry pointed out that it would have been misleading for the advert to have not included Jerusalem in a four-day itinerary of Israel, which is how the picture was represented.


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Federal Appeals Court Hears Arguments in Six Same Sex Marriage Cases
Posted: 08/07/2014 - A federal appeals court is hearing arguments in six same sex marriage cases from four states.
Posted: 08/07/2014 - A federal appeals court is hearing arguments in six same sex marriage cases from four states.
Updated: 08/07/2014 - President Obama is expected to sign the Veterans Affairs Reform Bill Thursday.
Updated: 08/07/2014 - AL.com -- Under fire from Congress over high cost and low returns, the Army National Guard announced Wednesday it is ending its NASCAR Sprint Cup Series sponsorship of Dale Earnhardt Jr.
Updated: 08/07/2014 - Montgomery, Ala. -With school starting in school systems throughout Alabama, motorists are cautioned to be especially careful as they travel along Alabama's streets and roads as school buses will again be on the road loading and unloading students across the state of Alabama.
Updated: 08/07/2014 - The Dothan Police Department needs your help locating two men for unrelated crimes.
Updated: 08/07/2014 - Abbeville, AL - Beginning Monday, August 11, 2014 a section of County Road 97 between Highland Park and the Hardridge Creek bridge will be closed to all traffic for road repair.
Updated: 08/06/2014 - Some call it trash - "Somebody threw this away and it had one broken arm on it," Butch Anthony said. But one man's trash - "Somebody caught that fish right there and there's a CD stuck on its nose," Anthony said.
Updated: 08/06/2014 - Since Monday, a transport bus, a Subway restaurant and a resident were all hit by a drive-by bb-gun shooter in Daleville.
Updated: 08/06/2014 - The former Geneva County Coroner was charged with impersonating a civil servant after routine traffic stop.
Updated: 08/06/2014 - Enterprise, Ala. -- Motorists traveling along S. Main Street in Enterprise for the past few days have been greeted by a new flowering display in what some might consider an unexpected location.
Posted: 08/06/2014 - ALBANY, Ga. (AP) -- A federal food safety inspector who investigated a deadly salmonella outbreak linked to a Georgia peanut plant says the company was "not fit to produce products for human consumption."
Updated: 08/06/2014 - Tallahassee, Fla. – Today, Governor Rick Scott announced two appointments to the Early Learning Coalition of Okaloosa and Walton Counties.
Updated: 08/06/2014 - (MONTGOMERY)— Attorney General Luther Strange announced the conviction of a Shelby County man for felony robbery and burglary violations.
Updated: 08/06/2014 - Troy University makes the Princeton Reviews list of Best Colleges and Universities in the Southeast.
Updated: 08/06/2014 - Bowe Bergdahl, the U.S. soldier who in May was traded for five Taliban operatives held at the Guantanamo, Cuba, detention center, will be question by Army investigators Wednesday.
Posted: 08/06/2014 - Montgomery, Alabama – Former Wilcox County Deputy Greg Barge, 45, from Camden, Alabama, was sentenced yesterday for attempting to possess with the intent to distribute cocaine powder, and using a firearm in furtherance of a drug trafficking crime, announced George L. Beck, Jr., United States Attorney for the Middle District of Alabama.
Updated: 08/06/2014 - 45,000 people are commemorating the 69th Anniversary of the atomic bombing of Hiroshima
Updated: 08/06/2014 - The World Health Organization says 932 people have died in the Ebola outbreak in West Africa. Saudi Arabia has also announced the death of one person with Ebola-like symptoms.
Updated: 08/06/2014 - Around 1,200 Dothan City School employees gather for the annual institute meeting at Wiregrass Church.
Posted: 08/06/2014 - The Dothan Education Foundation announces their teacher grant program recipients.
Updated: 08/06/2014 - Six Tuskegee University students are stuck in Africa because of the Ebola outbreak.
Updated: 08/06/2014 - New Brockton is cracking down on eyesores. Town council members voted to spend $2,500 to remove an old convenience store.
Updated: 08/06/2014 - A handful of roads in the Circle City may feel less bumpy in coming months.
Posted: 08/05/2014 - Some Carroll High School students went back to class on Tuesday...with their parents.
Posted: 08/05/2014 - Daleville residents enjoyed a night of fun to keep their community safe.


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Will poll bust for departing Coastal Carolina?
(STATS) - Now you see, now you don't. That's the scenario with the Coastal Carolina Chanticleers, who are playing their final season in the FCS as an independent while they transition to the FBS level next season. Officially, Coastal is already a member of the Sun Belt Conference - having joined on July 1, just one day after the Chanticleers' baseball team won the College World...
Via FOX Sports
18 days ago | Discuss
TCU uses full page ad to honor Coastal Carolina, which beat Frogs in College World Series
En route to winning the College World Series on June 30 to claim its first national championship in program history, the Coastal Carolina Chanticleers had to defeat TCU in a best-of-three series. The Chanticleers won the series 2-0 and went on to best Arizona 2-1 in the finals.The loss stung for the Horned Frogs, which has appeared in the past three College World Series and is still...
Via FOX Sports
24 days ago | Discuss
Coastal Carolina goes nuts celebrating CWS
Coastal Carolina did the seemingly-impossible on Thursday and defeated Arizona 4-3 in a thrilling game to win the College World Series. It was the first time the Chanticleers have ever won in their school's history, which dates back to 1954. And what a run it was. In the regional, they were losing to NC State and down to their final out, but came back to win. They then went 6-2...
Via The Sports Daily
29 days ago | Discuss
College World Series 2016: Coastal Carolina wins first national title
The 2016 College World Series championship series has certainly been a strange one as Wednesday night's Game 3 was pushed to Thursday afternoon due to multiple weather delays. The Coastal Carolina Chanticleers used those rain delays and held off the Arizona Wildcats to win their first national championship in school history.
Via Campus Sports Net
June 30, 2016 | Discuss
NCAA baseball tournament 2016: Coastal Carolina advances to Super Regional
It has been a strange ride for the Coastal Carolina Chanticleers this postseason, but the second seed in the Raleigh Regional has achieved something big by making it to the Super Regional. They finished their run to the second round with a comeback win over the NC State Wolfpack on Tuesday. COASTAL CAROLINA IS ON TO SUPER REGIONALS! The Raleigh Regional title belongs to the Chanticleers...
Via Campus Sports Net
June 07, 2016 | Discuss
Coastal Carolina to play Arkansas in 2017
The Coastal Carolina Chanticleers will be joining the Sun Belt in 2017, making a move from the FCS into the FBS in the process. They will certainly be getting a warm welcome as they will travel to face the Arkansas Razorbacks in their first season. The Razorbacks announced two non-conference opponents for the 2017 season as they will host the Chanticleers and the TCU Horned Frogs...
Via Campus Sports Net
April 12, 2016 | Discuss
Winthrop Closes Out Rivalry In Style, Remains Tied Atop Big South
Winthrop guard Keon Johnson (5) drains a free throw. The Eagles were perfect from the line in 20 attempts. (Brian Wilmer/CHD)Brian WilmerFollow @sportsmatters ROCK HILL, S.C. – Winthrop coach Pat Kelsey has frequently referred to sophomore forward Xavier Cooks as his team's "barometer", meaning that as he goes, the team goes. Coastal Carolina coach Cliff Ellis called...
Via College Hoops Digest
February 19, 2016 | Discuss
Coastal Carolina to play at UCLA in 2023
The Coastal Carolina Chanticleers will be moving up to the FBS next season as they will be joining the Sun Belt Conference. It looks like they are already getting big games scheduled for the distant future. According to FBSSchedules, the Chanticleers have scheduled to play on the road agaisnt the UCLA Bruins on September 16, 2023. The Chanticleers will receive $1.1 million as part...
Via Campus Sports Net
December 23, 2015 | Discuss
Coastal Carolina sees US flag raised at Embassy in Cuba
For the first time in fifty years, the flag of the United States of America is once again flying at the Embassy in Cuba. Last week, the flag was raised in a special ceremony in Havana by the same three Marines that lowered it in 1961 who vowed to be the ones to raise it again if that were to ever happen. According to College Basketball Talk, the Coastal Carolina Chanticleers' basketball...
Via Campus Sports Net
August 19, 2015 | Discuss
Coastal Carolina Claims Back-to-Back Big South Tournament Championships - 2015 VisitMyrtleBeach.com Big South Championships
Coastal Carolina wins the 2015 VisitMyrtleBeach.com Big South Championships. (bigsouthsports.com)By Brian Wilmer@sportsmattersCONWAY, S.C. -- Charleston Southern coach Barclay Radebaugh spoke a familiar refrain about the quality of the Big South Conference after his team's loss to Longwood tournament quarterfinals on Friday, stating that the conference was "the best it's...
Via College Hoops Digest
March 08, 2015 | Discuss
Coastal Carolina Holds Off Late Gardner-Webb Charge To Reach Big South Championship - 2015 VisitMyrtleBeach.com Big South Championships
Coastal Carolina forward Badou Diagne paced the Chanticleers in a semifinal victory. (goccusports.com)By Brian Wilmer@sportsmattersCONWAY, S.C. -- The destination was perfectly fine for Cliff Ellis' Coastal Carolina Chanticleers on Saturday. It was the journey that gave his team some concern.Both Gardner-Webb and Coastal Carolina had late nights in Friday's semifinals, with...
Via College Hoops Digest
March 07, 2015 | Discuss
Coastal Carolina Defeats UNC Asheville In Quartefinal Nightcap - 2015 VisitMyrtleBeach.com Big South Championships
Elijah Wilson scored a career high in Friday's victory. (goccusports.com)By Brian Wilmer@sportsmattersCONWAY, S.C. -- After star Asheville guard Andrew Rowsey compiled 34 points against Liberty in Wednesday's Big South Championships first-round action, it was a fair question to wonder who would stop him -- and how it would be possible. Playing in their first game of the tournament...
Via College Hoops Digest
March 07, 2015 | Discuss
Coastal Carolina planning to install teal-colored field
By Dan Lyons It took a little while to really catch on, but a bunch of schools are hopping on the colored field bandwagon that is famously driven by Boise State Last year, Eastern Michigan put in a gray field that has received mixed reviews. Now, the Coastal Carolina Chanticleers are jumping in on the trend with a teal field. The program released a rendering of the field on Twitter...
Via College Spun
January 24, 2015 | Discuss
NFL Scouts May Want to Watch Coastal Carolina's Devin Brown Return This Kick For a Touchdown
The only FBS game on Saturday is the Army-Navy game, but the FCS playoffs are still being played as well. Three-time defending champ North Dakota State has its hands full with the Coastal Carolina Chanticleers, but ended up pulling out a win, 39-32.While the Bisons got the win the highlight of the day belongs to this guy:@SBNationGIF/TwitterRead more »
Via FatManWriting
December 13, 2014 | Discuss
Coastal Carolina Chanticleers Are the No. 1 FCS Team (because they don't play like cats?)
Typically not much of the college football loving world pays a lot of attention to the FCS ranks unless they are upsetting an FBS team or there isn't anything else on television. Fact is these guys work just as hard and often play just as good if not better football than the big boys--just at another level.So when the Coastal Carolina Chanticleers were named the No, 1 team in the...
Via FatManWriting
November 11, 2014 | Discuss
Coastal Carolina Chanticleers to Install Teal-Colored Football Field?
The Coastal Carolina Chanticleers are a relatively young football program. As part of the FCS and a member of the Big South they are not exactly a team you hear about much unless they are playing one of the big boys in the FBS--like last year when South Carolina stomped them 70-10.But starting next season that is all going to change.@GoCCUSports/TwitterMove over Boise State...
Via FatManWriting
August 13, 2014 | Discuss
What Seed Did Coastal Carolina Get In The 2014 NCAA Tournament?
Seed: For the upcoming 2014 NCAA Tournament, the Coastal Carolina Chanticleers were given a 16-seed in the East-region. They are coached by Cliff Ellis and hail from the Big South Conference. You can view the entire 2014 NCAA Tournament field here in our official bracket. Where: They will open play against Virginia in Raleigh on Friday. Why: Coastal Carolina finished with an...
Via College Spun
March 16, 2014 | Discuss
Coastal Carolina Wins Big South Tourney, Warren Gillis Wins MVP
Coastal Carolina defeated Winthrop 76-61 to win the Big South Conference Tournament. Warren Gillis was named Tournament MVP and was joined on the All-Tournament Team by Josh Cameron, Keon Moore, D.J. Cunningham and Jerome Hill
Coastal Carolina ends 21-year NCAA drought
Warren Gillis had 22 points and Josh Cameron added 19 to lift Coastal Carolina to the Big South Conference tournament title and its first NCAA trip in 21 years with a 76-61 victory over Winthrop on Sunday.
Via Philly Sports Live
March 09, 2014 | Discuss
Coastal Carolina rallies past VMI 83-78
Elijah Wilson scored 26 points and grabbed four rebounds to lead Coastal Carolina in an 83-78 comeback win over Virginia Military on Wednesday night. Warren Gillis added 16 points and five rebounds for the Chanticleers (15-10, 8-3 Big South). Josh Cameron scored 15 points and Badou Diagne had 12. VMI led 20-9 at 13:39 in the first half and went into the break with a 50-36 advantage...
Via Associated Press
February 12, 2014 | Discuss
Coastal Carolina beats CCSU 86-67
Elijah Wilson scored 19 points as Coastal Carolina beat Central Connecticut State 86-67 on Friday night. Coastal Carolina (7-7) dominated the paint as Michel Enanga, Badou Diagne each recorded a double-double with points and rebounds - 13 and 10, 10 and 13, respectively. The Chanticleers held a 48-31 rebounding advantage, including 20 on the offensive end. CCSU (4-9) was paced...
Via Associated Press
January 03, 2014 | Discuss
Cameron lifts Coastal Carolina over CCSU 65-62
Josh Cameron made a four-point play with 22 seconds left to give Coastal Carolina the lead for good in a 65-62 victory at Central Connecticut State on Saturday. Coastal Carolina (6-6) trailed 62-59 when Cameron made a 3-pointer and the foul shot to put the Chanticleers up 63-62. CCSU (3-8) had a chance to go back ahead but Kyle Vinales missed a 3-pointer with 6 seconds to go and...
Via Associated Press
December 21, 2013 | Discuss
Coastal Carolina dumps Reinhardt 118-82
Badou Diagne scored a career-high 26 points to lead seven players in double figures as Coastal Carolina routed Reinhardt 118-82 on Saturday night. Warren Gillis contributed 21 points, Elijah Wilson added 16, Michel Enanga had 14, Josh Cameron had 12 and Colton Ray-St. Cyr had 10 for the Chanticleers, who shot 67.7 percent from the field. El Hadji Ndieguene pulled in 12 of Coastal...
Via Associated Press
December 15, 2013 | Discuss
Coastal Carolina roars past NC Wesleyan, 89-54
Josh Cameron poured in 21 points and led six players into double figure scoring as Coastal Carolina used a big second half to cruise past North Carolina Weslyan, 89-54 Thursday night. The Chanticleers led by nine at intermission, 35-26, but outscored the Battling Bishops by 26 points over the final 20 minutes. All of Cameron's field goal attempts came from behind the 3-point line...
Via Associated Press
December 05, 2013 | Discuss
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I posted this on facebook but had to share it with all of you ladies as well. Hillary was talking to me this afternoon when all of the sudden Liam spit up on her. After getting spit up on she said, "mom Liam just puked boob juice all over me and that is SOOO wrong!" That girl just cracks me up. Hope everyone is having a good Thursday!
funny things our LO's say!
Mom to 3 amazing kids
hahahaha that's hilarious!!! Thanks for making me laugh!
Nicole - mama to J ('07), G ('09), G ('12) & A ('15)
that's fantastic...everyone at work started making me post a daily "mckenna-ism" because she just cracks you up. this week's have been:
1. me: "mckenna lets try swimming without your vest." mck: "mommy, i'm just a little girl and i haven't learned yet." me: "well, how am i supposed to teach you if you won't take your vest off?" mckenna: "but i'm just a little girl."
2. (after her 1st day of school) mckenna: "mommy, school is really hard work. i'm so tired, i need to take a nap."
3. (she's been a little constipated this week so pooping hasn't been too easy, this was after she pooped last night) mckenna: "mommy, i'm all done. now that wasn't so bad."
McKenna 7/07 Connor 2/09 Brennan 8/11
I love these!
Earlier in the week when we were at my parents' house, Everett was waiting not so patiently for everyone to finish dinner so he could get up from the table. My dad (who is losing his hearing) and my grandmother (who has lost her hearing) were attempting to communicate with each other and just ended up yelling across the table. Everett turned to my sister and said pathetically "Aunt Chrissy, can you please get me out of this crazy place?"
Boy 07/07, Girl 12/10, Boy 03/05/13 VBA2C!!!!
HAHA!!! These are so funny!
Today- we had to take our cat to the groomer- we thought they would have to shave her (we got her about 3 weeks ago, she is long hair, and wasn't brushed much!)- on our way back to pick her up Danika said "Well, maybe she wont be a cat anymore" WTF- because getting her groomed will change the species! It was so funny!
Last week we were at my dads for dinner. She looked at my step mom and said "you have bubblies too" (that is what she called boobies). My step mom started laughing and said yes. The I told the story how she asked my brother why he had bubblies. He told her "because uncle Ryan is too fat"- well DH started laughing at that. Danika walked right up to him and said "Daddy, don't laugh, you are fat too" I almost fell off my chair!
Brianne- WAHM to Danika 7/07 & Bryce 5/09
lol, I love silly stuff our kids say!
Yesterday I was having a loud discussion with my mother and Corbin came stomping up to me and said "Momma! You don't talk back to your momma like that!!" Dang kid, I can't ever catch a break with him ;)
This is a dream that I've had since lunch, and I'm not giving up on it now. -Michael Scott
Mama to Corbin- 7/07 & Oliver 2/15/10
Gavin told daddy the other day, "I'm small, but I'm wonderful!"
He recently told me, "I have a tiny, little, cute, little nose."
The other morning in the middle of a tantrum, DH tried to get him to come give him a hug. Gavin says in a whiny, crying voice, "But I WANT to watch the Today Show and have my breakfast!"
Mom to G 7/13/07, L 6/17/10, and R 10/9/13!
Visit my ETSY shop for affordable, custom pencil drawings from your favorite photos!
Emma has been QUITE the Back-Seat-Driver lately.............so on Wednesday we were in Manhattan. We got stuck in some traffic, and she could see that the light was Green, but we werent going anywhere.
So she goes, "Mama, its Green, lets GO!"
I said, "We cant, there is a Car in front of us"
Then she goes "MOVE ASK-HOLE!!!!!!"
Needless to say DH gave me a NASTY LOOK, and goes "See?? I told you, you have to watch what you say around her........."
Mommy to Sebastian (13yrs), Emma (7yrs), Sarah (5yrs), and Nico (3yrs)
Awe - this is such a fun age to hear the things they come up with. I got this great book called "My quotable kid" where you journal all the funny things they say. Here are some of my favorites:
Those ants think I'm their lunch!
Mama, your toes are pink and my toes are Khaki.
Hey, that man forgot to put his hair on today.
Mom to Carter Pierce (7/15/07) and Brandon Bennett (2/11/11)
Not long ago Lavery said, "Mommy, you have big boobies. I have small boobies. Daddy has medium boobies." Needless to say, I didn't repeat that to DH. lol
"I think the reward for conformity is everyone likes you but yourself." -Rita Mae Brown
Oh, and related to Jaime's post.... now Lavery is a HUGE fan of saying how stupid all the other drivers are. Thanks to me.
"I think the reward for conformity is everyone likes you but yourself." -Rita Mae Brown


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Roger Phillip Burnett
member for 10 years, 11 months, 9 days
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Roger Phillip Burnett
b:10-28-1942 m Nell Lowery
Parents Taft Burnett &
June Jarrett buried at
Rebels Creek Cemetery
Mitcell Co.NC. Deacon at Chesnee First Baptist Church, Chesnee,SC.
Served in the USAF.
Retired from Duke Energy
Corp.Welding Inspection,
NDE Inspector.
Sing Baritone with The Carolina Quartet
family Qt.(Rebels Creek
Qt. for 10 yrs.
Masters Singers 17 years.
The Melody Masters Qt. organized about 1948.These 4 men have been together since 1992, check it out on www.youtube.com.) We recently changed the name of the Quartet to "Carolina Quartet".
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Sonya Webb
RE: Siblings
Thank you!
Added by
Sonya Webb
on Jul 17, 2016 3:56 PM
Deborah Strum
John Moccasin Green and son, James
I think that your work has led me to my ancestor, John Moccasin Green. However, you show that his son James died in 1856; when,in 1857, James, who lived in Haywood County, wrote a letter for John's Rev. pension citing himself as John's son.
James' daughter Charlotte "Lettie" married my third-great grandfather, Moses Brown, who died in the Civil War. They had a number of children including my 2nd ggrandfather, Isaac Brown.
Added by
Deborah Strum
on Jul 08, 2016 8:38 PM
Tee Tee
Gilbert Henry Thomas 81839050
It states on Find A Grave that Thomas,81 of Roses Branch Road, died Friday, Dec.27,2002. He was the son of the late LeRoy and Laura Woody Thomas.
Then you have Jobe Thomas under parents. I can assure you Jobe Thomas was long passed away before this Gilbert Thomas came along.
Please make corrections.
Thank you so much for all your work,
Added by
Tee Tee
on Jun 19, 2016 12:13 PM
Phil Mullen
Jonathan Barron Will
I don't have the complete will of Jonathan Barron just an excerpt from it naming his children. It appears on the memorials of Catherine Carmilla Weatherman's brothers, viz: Henry Phifer Barron 26966023, Francis Marion Barron 28799042 and Pleasant Wilson Barron 64036631. You may want to add it to Caherine's memorial as well.
Thanks for your kind comments.
Phil Mullen
Added by
Phil Mullen
on Jun 04, 2016 9:14 AM
Phil Mullen
Old Lady Weatherman
Nice work you and others did researching "Old Lady Weatherman". Weatherman is the correct spelling. She is listed as a child in the will of her father Jonathan Barron, refer to memorial 64036631 to see the will.
Phil Mullen
Added by
Phil Mullen
on May 31, 2016 9:50 AM
Frances Hall
Benjamin Willis, Jr.
Do you know who has proof for this information on "Find A Grave"? I would like to correspond with them. After searching for many years to find proof of this family,I am so excited to see this. Almost too good to be true. Whoever did this deserves a medal, because this has been a very difficult family to prove.
Frances Hall
Added by
Frances Hall
on May 28, 2016 6:27 PM
Jeff Lovelace
Please see this memorial: 161804973.
Added by
Jeff Lovelace
on May 06, 2016 12:14 PM
Barb Dowden
Lieut Jackson Stewart
Memorial # 62847675: What proved to do have that he mit be buried in Greene Cemetery, for a marker to be place here and his son Jasper. I have proved that a Vet. marker was order for Jackson the 20 Nov 1864 for Burleson Cemetery (Find A Grave now call it Loafers Glory Cemetery); if I am correct these marker that was put in Greene Cem , should have been put in Burleson Cem now Loafers Glory Cemetery. Check it out and see what you think, let me known, for I believe he buried in Burleson Cemetery(now LGC)not in Greene, thou I could be wrong. Thanks Barb
Added by
Barb Dowden
on Apr 29, 2016 10:38 PM
Barb Dowden
Infant Bartlett
Find A Grave # 67398559 and 68017116 are one and the same Memorial, just though to bring it to your attention, Thanks, Barb
Added by
Barb Dowden
on Apr 27, 2016 6:18 PM
regarding RE McKinney, MB McKinney, MaxineJOllis
Please transfer 62633882,62632374,67344173 to me.I am related to all three and would like to manage them. Thank you sincerely for all you have done and a response is appreciated. God bless.
Added by
on Apr 20, 2016 6:34 PM
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The system crash dialog is annoying me, how can I turn it off? I'd also like to know how to turn it back on just in case I need it to report a problem.
You need to edit
Graphical: Open a terminal with (CTRL+ALT+T) and type this:
and then push ENTER. You password is being typed, but will not display as dots.
Command line:
A file editor is now open. Change enabled from "0" to a "1" so it looks like this:
To turn it off make it:
Now save your changes and close the file editor. Apport will now no longer start at boot. If you want to turn it off immediately without rebooting, run
You can also use
See also :
The above script should stop apport, then take a backup of its configuration file, disable apport on boot, and lastly moves the backup to your home directory.
On newer versions of ubuntu (15.04+)
To stop the service:
To disable the service at startup:
To check the status of the service:
Finally, you can also prevent the startup of a systemd service by masking it. The service will not be able to start (even manually) unless unmasked.
This should create symlink from /etc/systemd/system/apport.service to /dev/null. fedoraproject.org
Don't disable apport. One of these days, you might have a sequence of crashes and never know it except for system bad behavior or some application-specific symptom.
Suggested procedure:
The repetitive crash pop-up behavior should now be gone. Also, the crash reports that you saved might be valuable in reporting a bug to launchpad.
Since there's a bug in apport that breaks standard unix command line debugging, and this bug has been known since 2007 (I'm going to buy it a cake when it turns 10) (see https://bugs.launchpad.net/ubuntu/+source/apport/+bug/160999) turning it off is far and away the best thing to do if you are trying to fix your own code.


text:
The Dothan American All-Star team won the AAA Distract tournament with an undefeated 5-0 record.
That earned them the right to play for a state title.
Before they head to Fairhope, Alabama to compete, they're holding a camp to raise money for their trip.
The All-Stars and their coaches invite all baseball players, ten and under, for a day of learning.
This Saturday, July 12th the players will give lessons on hitting, pitching, defense and running the bases.
Coaches say the best part of the camp is that players will learn first hand from the team.
The camp costs $25 per player. Gatorade and snacks will available for all players.
Registration beings at 8 a-m on Saturday at Doug Tew park.
For more information contact:
Michael Smith- 701-6189
Chris Sulllivan 405-1340


text:
The New Human Capital Strategy: Improving the Value of Your Most Important Investment-Year After YearIt is often said that the only true source of sustained competitive advantage is people. But what does that mean and how can this be measured and managed? How many organizations know whether their human capital outperforms their competitors, or even whether it improves year-over-year? And what is the strategy for continually improving that performance? The New Human Capital Strategy is a roadmap
From Difficult to Disturbed: Understanding and Managing Dysfunctional EmployeesThe book contains down-to-earth solutions for dealing with: Personality Types including avoidant, dependent, histrionic, narcissistic, or antisocial workers; Common People Problems such as unproductive, angry, uncooperative, or chronic problem employees; and Mental Disorders such as depression, bipolar disorder, schizophrenia, anxiety and panic disorders, as well as alcohol and substance abuse.
How to Sell Anything to Anybody"Salesmen are made, not born. If I did it, you can do it." -- Joe Girard In his fifteen-year selling career, author Joe Girard sold 13,001 cars, a Guinness World Record. He didn't have a degree from an Ivy League school -- instead, he learned by being in the trenches every day that nothing replaces old-fashioned salesmanship. He insists that by building on basic principles of trust and hard work
Helping People Through Corporate ChangeA practical, hands-on guide to helping employees through the often painful process of organizational change--whether from technological innovation, limited resources, or new management. Based on years of experience, this field-tested book will teach the crucial skills and strategies to help managers deal with their co-workers' reactions to change; minimize the disruptions in productivity,
The Principles of Winning at Sports Applied to Winning in BusinessSport Psychologist Dr. Jim Loehr and marketing consultant Peter Mclaughlin outline techniques that can be used to achieve the mental toughness displayed by professional athletes. They take these techniques--including visualization, motivation, performance ritual, breath control, and more--and demonstrate how they can be effectively applied in the business world.Mental toughness allows anyone to
50 Activities for Team Building, Volume 2Create high performance teams with these collections of fully reproducible activities. All the activities employ the principle of "learning by doing" to ensure that the skills the participants learn can be applied to real work situations. Volume 2 offers 50 additional activities for team development and provides practice on cooperation, balancing roles, and defining team objectives and goals.
What Must Go Right/What Can Go Wrong
SAGE Series in Interpersonal Communication (Book 10)What are the secrets of successful teams? Why do some teams achieve remarkable success while others fail or are consigned to mediocrity? To find the answers, Larson and LaFasto interviewed a wide range of teams, including the space shuttle Challenger investigation team and executive management teams, and discovered a surprising consistency in the characteristics of effective teams. The authors
$73.59This cross-disciplinary text is designed to appeal to a diversity of social science scholars. The central focus is on new ways of viewing the career, or how working lives unfold over time. Fresh views from psychology, social psychology, sociology, anthropology, organization theory, economics, and political science are among those represented in the twenty-five chapter anthology. The design of the
$13.29How a business replaces its chief executive often determines that firm's future. If a business does not effectively manage the transfer of power, utter turmoil can result, with profound implications not only for the CEO, but also for the other employees, the shareholders, and the community at large. Filled with inside stories from corporate boardrooms and fresh conceptual perspectives, The Hero's
$8.69The authors use excerpts from hundreds of actual business documents to test your writing skills and illustrate and correct the most common grammatical pitfalls executives must avoid. All examples are taken from real-life business and professional situations--actual memos, articles, annual reports and transcripts of national news reports. Short tests help you master the forty essential guidelines
Unleashing the Power of Your Total Workforce by Managing DiversityThe ability to manage this diversity successfully has become a basic strategy for corporate survival. Beyond Race and Gender supplies a sorely needed Action Plan, extensive case studies, and a series of tough questions and answers to get readers thinking deeply about what elements are blocking the full use of the human talent available. In this visionary work, R. Roosevelt Thomas, Jr., rouses


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EMEA Suppliers: Visit http://cbre.globalrms.com/emea/ to enroll or renew in the CBRE Global Supplier Screening Program.
Welcome to the CBRE Supplier Screening Program
In an effort to strengthen our company's risk management program and ensure that we are in compliance with federally mandated laws and regulations, CBRE/GWS has implemented a Global Supplier Screening and Certificate of Insurance Management Program as part of our risk management policy.
We now require all current or potential service suppliers who conduct business with our company to enroll and complete the screening process through our partner, Global Risk Management Solutions (GRMS), in order to become "approved suppliers" for CBRE.
CBRE's Supplier Screening Program is designed to streamline the collection and management of key information regarding the regulatory and compliance status as well as business operations of our suppliers and suppliers.
- All current and potential CBRE service suppliers must enroll in the Supplier Screening Program and pass CBRE's screening criteria
- There is a non-refundable $350 USD annual enrollment fee associated with the program for U.S. and Canadian suppliers.
- If applicable, an additional fee of $150 USD will be assessed for Third Party Oversight (TPO) screening.
- You will be able to view the results of the background screening and have the opportunity to correct any incorrect information.
- Failure to enroll and become compliant may result in the discontinuation of your company's services by your client.
- If you have previously enrolled with another CBRE client through this method, you must complete the COI screening process for that client account at no cost to be included as an approved supplier for this client. Be sure to use the same EIN number that was used in the previous enrollment. This is the key to eliminating duplication. You should have received a url to the client account website in a subsequent letter or email.
- Samples of the required documents and insurance limits can be found here.
- This screening program is designed primarily for suppliers with annual spend of $5,000 USD or greater.
Successful completion of this process will result in your company being added to the CBRE/GWS Approved Supplier List. This list will be available for all GWS accounts to view. In the near future, only suppliers included on this list will be eligible for work on GWS client accounts. This will provide your company with the visibility across GWS accounts that many of our suppliers have been requesting. Please be sure to keep the information on your GRMS account updated to insure we can contact the appropriate people and that our client account teams can determine your geographic availability and service capabilities.
How Do I Enroll
The enrollment process is web-based, easy, and available to you today. Start the process by clicking the button below. Once the enrollment form is completed, you will be presented with a payment screen. After your payment has been processed, you will receive a receipt via email and login credentials in a separate email within 24 hours.
If you have previously enrolled or believe that you have been contacted in error, please submit your request at http://www.GlobalRMS.com/removal and you will no longer be contacted.


text:
Thank you for visiting the Carolina Regional Volleyball Association, a proud member Region of USA Volleyball (Carolina Region/USAV). Our goal is to be your one-stop site for all information about volleyball in North Carolina. Look around, explore, and let us know how we can help you enjoy the sport!
Help support the Carolina Region while doing your regular online shopping! The Carolina Region is a part of the Amazonsmile foundation charitable giving program. Start your Amazon online shopping from the link below and Amazon will donate 0.50% of your eligible purchases to the region. There is no cost to you! Thank you for your support.
Carolina Region's Amazonsmile start page


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Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA.
Cells have evolved complex mechanisms to halt cell cycle progression in response to genomic insult, collectively termed the DNA damage checkpoint [1–3]. These checkpoint controls allow time for repair or bypass of the lesioned DNA, or, if the damage is too severe, to activate the apoptotic program [4–8]. The Phosphatidylinositol 3-kinase-like kinases ATM and ATR are central upstream regulators of the DNA damage checkpoint, phosphorylating consensus SQ or TQ motifs on downstream effectors, such as the kinases Chk1 and Chk2 and the transcriptional regulator p53 [1,9–27]. Chk1 and Chk2 kinases in turn phosphorylate substrates, including p53 and the Cdc25 phosphatases, which halt cell cycle progression through inhibition of cyclin dependent kinase activity [14,21,28–33].
In addition to the core DNA damage checkpoint pathway, hundreds of other proteins have been identified that participate in a more generalized DNA damage response (DDR) [3,34–36]. Proteomic analyses of DNA damage signaling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) have identified hundreds of candidate ATM/ATR substrates more highly phosphorylated in response to damage [37–40]. These studies, along with years of more focused biochemical analysis, emphasize the far-reaching effects of the DDR in affecting protein post-translational modification across many signaling pathways controlling diverse cellular functions.
In addition to studies of the DNA damage response, antibody-based proteomic methods have also been employed to study signaling areas, such as cancer, neurodegenerative disease, growth and development and infectious disease [37,39–52]. Other LC-MS-based proteomic methods of PTM analysis have employed sample fractionation and whole proteome analysis or global phosphopeptide enrichment methods, such as immobilized metal affinity chromatography (IMAC) with chelated iron or titanium dioxide beads [38,53–66]. Such fractionation and enrichment strategies, combined with the sensitivity and speed of current LC-MS/MS systems, allow identification of hundreds to thousands of post-translationally modified peptides in a single LC-MS/MS run. These methods suffer, however, from the fact that the peptides identified and quantified are randomly sampled by the instrument and tend to focus on the more abundant peptides present in a sample, while critical signaling may be mediated by proteins expressed at exquisitely low levels. The results from these proteomic studies can therefore lack the specificity necessary to probe signaling networks in enough detail to sufficiently understand the mechanism of action or cellular response to a particular perturbation.
A novel method for identification and quantification of post-translationally modified peptides, termed PTMScan Direct, was recently published [67]. This method enables focused study of peptides from proteins in the same signaling pathway or from the same protein type. Four PTMScan Direct reagents were previously designed that probed critical signaling nodes of many different pathways (the Multipathway Reagent), phosphorylation sites on kinases (the Ser/Thr Kinases Reagent and Tyr Kinases Reagent) and Phosphatidylinositol 3-kinase signaling networks (the Akt/PI3K Pathway Reagent). Each reagent allows quantification of hundreds of post-translationally modified peptides from proteins that reside in the desired signaling space in a single LC-MS/MS run. PTMScan Direct can be used on cell lines, tissues, xenografts or primary cells. The method has been validated for use in humans and mice, but could be extended to include any species with an available proteomic database (for peptide identification). This method therefore combines the specificity of traditional biochemical analysis with the speed, sensitivity and high number of endpoints inherent to LC-MS/MS analysis.
Two new PTMScan Direct Reagents have been formulated that target DNA damage and cell cycle pathways, as well as apoptotic and autophagolytic pathways (PTMScan Direct: DNA Damage/Cell Cycle and Apoptosis/Autophagy Reagents, respectively). Each PTMScan Direct reagent allows in-depth analysis of its targeted pathways by focusing the MS sampling on the immunoaffinity-enriched target peptides, eliminating the random sampling that can occur with traditional global enrichment strategies. Reagents were rigorously validated using both human samples, as well as mouse tissues, with only peptides that pass strict criteria (MS/MS score filtering; intensity filters; enrichment versus control filters; presence of a sequence targeted by the reagent, etc.) accepted as validated targets. The DNA Damage/Cell Cycle Reagent allows identification of 263 sites on 137 proteins, while the Apoptosis/Autophagy Reagent targets 175 sites of phosphorylation and caspase cleavage activation sites on 100 proteins.
These unique reagents have been used to study activation of the DNA damage checkpoint and induction of apoptosis in HeLa cells damaged with UV light. In response to UV damage of DNA, peptides from markers of checkpoint signaling (ATM, Chk1, Chk2), cell cycle arrest (p21), stress response (p38 MAPK, JNK) and apoptosis (caspase cleavage, cytochrome c) all changed in abundance in the PTMScan Direct studies. Changes to selected signaling nodes were confirmed by western blotting, highlighting the accuracy of the method. These reagents therefore provide detailed, reproducible analysis of hundreds of peptides from proteins involved in the response to DNA damage and damage-induced cell death in a single LC-MS/MS run and are powerful tools for LC-MS/MS-based proteomic analysis.
Two new PTMScan Direct reagents have been developed to study the DNA damage response and cell cycle progression, and apoptosis and autophagy pathways. Reagents were prepared with a focus on proteins and sites in curated pathways, such as those found at www.cellsignal.com (Cell Signaling Technology, Danvers, MA, USA. Both reagents were validated using a strategy outlined previously [67]. Briefly, reagents were used to immunoaffinity purify (IAP) peptides from a mixture of human cancer cell lines treated with pervanadate to artificially increase phosphopeptide levels ("CST Cell Line" in Tables S1–S4), as well as mouse liver, brain and embryo samples [67] (data not shown). Jurkat cells treated with pervanadate or etoposide were also run as validation samples (see Tables S1 and S2). To be considered validated, the peptides identified had to pass a number of filters, including score filtering of the MS/MS assignment, a minimum MS1 signal intensity filter (minimum 20,000 for MS1 peak height from XCalibur processing (v2.0.7; Thermo Fisher Scientific, Barrington, IL, USA)) and a higher abundance of the peptide in the PTMScan Direct Reagent IAP than in a control IAP using empty protein G beads. Candidate peptides were then limited to those originally targeted by the reagent or homologous to a target and peptides from proteins involved in the signaling area of interest. Complete lists of peptides validated using this strategy with accompanying MS/MS scoring metrics are available in Tables S1 and S2.
Proteins for which peptides can be identified using the DNA Damage/Cell Cycle Reagent are shown in Figure 1. Interactions are from the STRING database, and pathway maps were created in Cytoscape 2.8.1 [68,69]. The interaction map in Figure 1 is organized by protein function, with the core checkpoint proteins ATM, ATR, Chk1 and Chk2 in the center of the map highlighted by a red box. The reagent goes well beyond identification of canonical checkpoint proteins to cover other DNA damage response proteins, including those involved in stress response, DNA repair, DNA replication, transcriptional regulation, adhesion and cytoskeletal organization, and cell cycle control. Overall, peptides representing 263 sites on 137 proteins were validated for the DNA Damage/Cell Cycle Reagent. A full list of proteins and sites identified using the reagent is available in Table S1.
The interaction map in Figure 2 shows proteins for which peptides can be identified using the PTMScan Direct: Apoptosis/Autophagy Reagent. As in Figure 1, interactions are from the STRING database imported into Cytoscape 2.8.1. The reagent targets peptides from a number of autophagy-specific factors, such as ATG proteins, kinases, including Akt, Erk, PKC and IKK, transcription/translation proteins, such as IkBa, NFkB, myc and Jun, and a large number of apoptotic proteins, including caspases, BAD, Bcl-xL, cytochrome c and FAS and TNF receptors. The caspase peptides targeted include sites of caspase cleavage and activation, such as Asp175 of caspase 3, Asp179 of caspase 6, Asp198 of caspase 7, Asp374 of caspase 8 and Asp315 of caspase 9. All peptides validated as targets of the reagent are shown in Table S2.
The two reagents were used to assess activation of the DNA damage response and onset of apoptosis in HeLa cells treated with 50 mJ/cm2 UV light and harvested 2 h post-treatment. These treatment conditions are effective in causing significant DNA damage and activation of checkpoint signaling [40]. Cells were harvested, digested and immunoprecipitated with each of the PTMScan Direct reagents in parallel as outlined in Figure 3. Replicate immunoaffinity purifications were performed with each reagent, and each sample was injected on the LC-MS/MS in duplicate. The complete data tables including scoring of the MS/MS assignments and label-free quantification are available as Table S3 for the DNA Damage/Cell Cycle Reagent and Table S4 for the Apoptosis/Autophagy Reagent.
The interaction map for the DNA Damage/Cell Cycle Reagent shown in Figure 1 is replicated in Figure 4A, color-coded by changes in peptide abundance in response to UV damage of HeLa cells. Proteins from which peptides were identified that increased in abundance at least 2.5-fold with UV damage are shown in green, while red signifies decreases in abundance of at least 2.5-fold. Selected peptides from proteins highlighted in Figure 4A with a red box are shown in greater detail in Figure 4B. The highlighted peptides are derived from the stress-responsive MAP kinases p38a, p38g, JNK1, JNK2, JNK3 and the upstream MAP kinase-kinase MKK4. Each of these phosphopeptides increased in abundance with UV damage, from 3.7- to 35.0-fold. Western blots in Figure 4C confirmed that, while there was no change in total levels of p38 or JNK, their phosphorylation at critical activation loop residues [71–75] did in fact increase, consistent with the PTMScan Direct data.
The blue box in Figure 4A highlights the core checkpoint proteins ATM, ATR, Chk1 and Chk2. Unmodified and phosphorylated peptides from these proteins are shown in Figure 4D (unmodified peptides are those with "–" in the Site column of Figure 4D). In each case, the unmodified peptides did not change in abundance with UV damage (1.3- to 1.0-fold change), while the phosphopeptides (ATM Ser1981, Chk1 Ser317 and Ser345, and Chk2 Ser379) by contrast all increased in abundance with UV damage (2.9- to 8.1-fold). The ATR peptide phosphorylated at Ser428 did not show an increase with UV damage in these cells (1.1-fold change). Western blots in Figure 4E are again consistent with the PTMScan Direct data, showing no change in protein level as assessed by the total ATM and ATR antibodies, an increase in signal with the ATM S1981 phospho-specific antibody and no change in signal with the ATR S428 phospho-specific antibody.
Other checkpoint proteins were also affected by damage, including the ATM/ATR family member DNA-PK, from which a Ser3205-phosphorylated peptide was identified that increased 6.2-fold with UV treatment. The DNA damage-associated histone, histone H2AX [76,77], was identified singly phosphorylated at Thr137 and dually phosphorylated at Thr137/Ser140, and both the singly and doubly-phosphorylated peptides increased with damage (4.9-fold increase and 5.1-fold increase, respectively). p21cip1 is a tumor suppressor protein that blocks cell cycle progression through inhibition of cyclin-dependent kinase (Cdk) and PCNA activity [78–81]. Unmodified peptides from the p21cip1 protein, as well as a Ser130 phosphopeptide, were identified and all decreased with UV treatment, from -4.8- to -10.8-fold.
The quantitative data collected using the Apoptosis/Autophagy Reagent is shown mapped onto the pathway diagram for the reagent in Figure 5A. As in Figure 4A, proteins with peptides that increased in abundance over 2.5-fold with UV treatment are shown in green, while decreases of at least 2.5-fold are shown in red. Selected caspase peptides highlighted in Figure 5A with a green box are shown in detail in Figure 5B. Peptides containing the cleavage and activation sites increase with damage for caspase 3 (3.1-fold), caspase 6 (2.8-fold) and caspase 7 (2.7-fold). The peptide derived from a cleaved form of caspase 8 increased slightly (2.3-fold), as did the cleaved peptide for caspase 9 (1.9-fold). None of the unmodified/uncleaved caspase peptides changed to this level with damage (max fold change = −1.4). Phosphorylated caspase peptides were also identified, and some increased with UV treatment including caspase 7 Ser199 (18.6-fold) and caspase 9 Ser302 and Ser307 (3.4-fold change for both). A phosphorylated caspase 9 peptide cleaved at Asp315 was also identified and increased significantly with UV damage (14.1-fold). Unlike the other phosphorylated caspase peptides, the S176-phosphorylated caspase 3 peptide did not change with damage (1.1-fold change). Western blots using caspase antibodies (Figure 5C) confirmed the PTMScan Direct results, with no change in full-length caspase 3, 7 or 8 protein levels, but a modest increase in the cleaved forms of caspase 3 and 7, and a slight increase in the cleaved form of caspase 8.
Figure 5A also highlights changes to other proteins beyond the caspases. c-Jun and JunD phosphopeptides both increased with UV treatment. Ser58, Thr62, Ser63 and Ser73-containing peptides for c-Jun increased in abundance 3.7- to 22.6-fold. A JunD peptide phosphorylated at Ser100 increased 9.7-fold with damage. Unmodified peptides from the cytochrome C subunit CYC1 increased in abundance with UV damage, from 2.4- to 8.4-fold. An IkBa Ser32 phosphopeptide decreased 2.5-fold with UV treatment, while a triply phosphorylated IkBa peptide (Ser32, Ser36, Tyr42) decreased very slightly with UV (-1.4-fold). Other changes highlighted in Figure 5A were not consistent across all peptides, for example DNA-PK is highlighted in green, though only one unmodified peptide changed >2.5-fold, while 29 other unmodified peptides from DNA-PK did not. Similarly, only two out of 16 LDH-A peptides increased >2.5-fold with damage (see Table S4).
Figures 1 and and22 highlight the capabilities of the new PTMScan Direct reagents to assess particular signaling pathways in detail. The DNA Damage/Cell Cycle Reagent provides an in-depth survey of proteins responsible for various functions in both normal cell cycle progression (DNA replication proteins, transcriptional regulators, cyclin dependent kinases and associated cyclins, etc.) as well as proteins that function in regulating the DNA damage response (checkpoint proteins, such as ATM, ATR, Chk1, Chk2 and ATRIP, stress responsive kinases, such as p38 MAPK and JNK, DNA Repair/Recombination factors, etc.). The Apoptosis/Autophagy Reagent allows the study of autophagolytic pathway markers, such as AKT, PI3K, PKC and ATG proteins, as well as apoptotic proteins, such as Fas and TNF receptors, caspases, BAD and Bcl proteins, cytochrome c and c-Jun. The diversity of proteins that can be monitored with these reagents make them powerful tools in the study of many signaling areas, including the response to UV damage of DNA. Further information about both reagents, including complete lists of proteins/sites validated, as well as sample data, can be found at http://www.cellsignal.com/services/direct_overview.html (Cell Signaling Technology, Danvers, MA, USA).
The PTMScan Direct data in Figure 4 shows that the UV treatment of HeLa cells was sufficient to activate DNA damage checkpoint signaling, consistent with previous results [40]. The DNA Damage/Cell Cycle Reagent was designed to allow detection and quantification of both phosphorylated and unmodified peptides for the core DNA damage checkpoint proteins ATM, ATR, Chk1 and Chk2. This allowed differentiation between increased phosphorylation of a static level of each protein versus an increase in the total protein level, with no change in the fraction modified. The observation that there was no change in abundance of any of the unmodified peptides indicates that levels of these proteins remained consistent, while their phosphorylation states changed. Phosphorylation of ATM at Ser1981 increased with damage, as did phosphorylation at multiple sites on the effector kinases Chk 1 (Ser308, Ser317, Ser345) and Chk2 (S379).
Interestingly, the phosphopeptide containing Ser428 of ATR did not increase in abundance with damage, even though ATR is known to respond to UV damage and Ser428 phosphorylation has increased with UV treatment in other systems (www.cellsignal.com, Cell Signaling Technology, Danvers, MA, USA). The PTMScan Direct data, however, were consistent with the western blotting results in Figure 4E, where no change in signal was observed −/+ UV treatment using the ATR Ser428 antibody. This suggests the lack of response was not due to any technical limitations of PTMScan Direct, but rather a real reflection of the activation state of the samples. It should also be noted that, while Ser1981 of ATM is a critical site of phosphorylation and activation of ATM [82], Ser428 of ATR does not function in an analogous manner. There is another site on ATR, Thr1989, that is postulated to fulfill a similar role to Ser1981 of ATM [83–85]. Thus, no change in Ser428 phosphorylation may not indicate a lack of ATR activity in the study. The phosphorylation of Chk1 at ATR-dependent sites [86] also suggests ATR may be active, but not robustly phosphorylated, at Ser428 at the time point the cells were harvested.
There are other lines of evidence to indicate activation of the DNA damage checkpoint beyond ATM/ATR-Chk1/Chk2 signaling in this study. DNA-PK phosphorylation increased in response to UV damage at Ser3205. This is a site previously identified and responsive to DNA damage [87,88]. Activation of DNA-PK can affect DNA repair activities, as well as checkpoint signaling [89,90], and phosphorylation of this site may affect the activity of DNA-PK in these processes. Histone H2AX is a histone variant that is recruited to sites of DNA damage and phosphorylated, and it is thought to act as a scaffold for the binding of other checkpoint proteins at the DNA lesion [76]. H2AX phosphorylation increased with UV damage at Thr137 and Ser140, sites known to be phosphorylated by DNA-PK, JNK and p38 MAPK [91–95], all of which were phosphorylated and activated by UV treatment in this study. A decrease in p21cip1 peptides (both unmodified and phosphorylated at Ser130) was observed upon DNA damage. Changes to all p21cip1 peptides suggest regulation at the protein level in response to UV, rather than specific changes to a phosphorylation site. Previous studies have suggested a p53-dependent increase in p21cip1 levels in response to DNA damage [78,96], the opposite of what is observed here. Numerous reports, however, have found a dose- and time point-dependent UV-induced decrease in p21 protein levels in a variety of cellular systems [5,97–100], consistent with the results of this study. A p53-dependent increase in p21cip1 level may only occur at later time points post-damage than the two-hour harvest time employed here.
In addition to activation of canonical DNA damage checkpoint signaling, the stress responsive MAP kinases p38 and JNK were also activated by UV treatment. These kinases were identified phosphorylated at their activation loop sites (Thr180/Tyr182 for p38, Thr183/Tyr185 for JNK), which correlate with kinase activity and an increase in phosphorylation in response to UV damage [71,73,101]. Western blotting in Figure 4C also confirmed that the changes observed in PTMScan Direct were due to changes in phosphorylation state, rather than total protein level for both p38 MAPK and JNK.
Figure 5 highlights the changes in peptide abundance observed using the Apoptosis/Autophagy Reagent. Most notable among these changes are the increased abundance of caspase peptides corresponding to cleaved, activated caspases. These increases were confirmed with the western blotting shown in Figure 5C, where the cleaved forms of caspases 3, 7 and 8 are more intense in the UV damaged sample than the control. It is also interesting to note that the phosphorylated forms of caspase 7 and caspase 9, but not caspase 3, increased with UV treatment. These phosphorylation events are not well characterized, but were previously identified in mitotic cells [102,103]. The biological impact of phosphorylation of caspases at residues close to their cleavage sites is not known, but may affect caspase activity, localization or protein-protein interactions.
Other peptides beyond those from caspases showed quantitative changes with UV treatment using the Apoptosis/Autophagy Reagent. Phosphopeptides from both c-Jun and JunD increased in abundance with UV damage. Ser63 and Ser73 of c-Jun and Ser100 of JunD are all JNK phosphorylation sites [72,73,104], and c-Jun is known to be induced by UV treatment [72,105,106]. As observed in Figure 4, JNK was activated by UV damage, providing a rationale for the increase in Jun phosphorylation. An IkBa peptide phosphorylated at Ser32 decreased with UV damage. Phosphorylation of IkBa increases upon UV damage [107–109], the opposite of what was observed in this study. UV damage-induced phosphorylation of IkBa, however, also leads to its ubiquitination and proteasomal degradation [110–112]. Thus, the decreased abundance of IkBa peptides in this study is likely due to a proteolysis-mediated decrease in IkBa protein levels.
Unmodified peptides from the cytochrome C subunit CYC1 also increased with UV treatment, though the reason for the increase is unknown. It is unlikely that it is due simply to release of CYC1 from mitochondria, as the lysis protocol used is known to free mitochondrial proteins (data not shown), and the related cytochrome C subunit CYCS does not show an increase in protein level with DNA damage. UV may therefore affect new transcription of CYC1 or interfere with its degradation, allowing accumulation in cells.
PTMScan Direct is adapted from the PhosphoScan method developed at Cell Signaling Technology [47]. The method and reagent validation strategy are previously described [67].
HCT-116, HeLa, NCI-H1703, NCI-H1299, Jurkat, T47D and A549 cells were from the American Type Culture Collection (Manassas, VA, USA). MKN-45 cells were from DSMZ (German Collection of Microorganisms and Cell Cultures). NCI-H3255 cells were from Dr. Lewis Cantley (Harvard Medical School, Boston, MA, USA). Etoposide treatment was for 6 h with 25 μM. Sodium pervanadate treatment was for 20 min with 50 ng/mL. Pervanadate treated cells were combined to create a cell line mix "CST Cell Line" in Tables S1–S4 to be run for validation of the reagents and as a control sample for all subsequent studies. Cells were cultured in recommended media supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37 °C with 5% CO2. UV treatment was performed in a Stratagene 2400 Stratalinker at 50 mJ/cm2. Cells were treated with the media and plate cover removed. Then, the media was re-added, and cells were incubated for 2 h prior to harvesting. Peptides from 10 mg total soluble protein were used for a single immunoprecipitation, corresponding to roughly 1 × 108 cells. Preparation of tryptic peptides from cellular lysates for immunoaffinity purification (IAP) was performed as previously described [67].
Immunoprecipitated peptides were resuspended in 0.125% formic acid and separated on a reversed-phase C18 column (75 mm ID × 10 cm) packed into a PicoTip emitter (~8 mm ID) with Magic C18 AQ (100 Å × 5 mm). Peptides were eluted using a 72 min linear gradient of acetonitrile in 0.125% formic acid delivered at 280 nL/min. Tandem mass spectra were collected in a data-dependent manner with an LTQ-Orbitrap Velos or LTQ-Orbitrap Elite mass spectrometer running XCalibur 2.0.7 SP1 using a top-twenty MS/MS method, a dynamic repeat count of one and a repeat duration of 30 s. Real time recalibration of mass error was performed using lock mass with a singly charged polysiloxane ion m/z = 371.101237 [113]. MS/MS spectra were evaluated using SEQUEST 3G and the SORCERER 2 platform (v4.0, Sage-N Research, Milpitas, CA, USA) [70]. Human samples were searched against the NCBI homo sapiens FASTA database, updated on 27 June 2011. Results were filtered using a mass accuracy of +/− 50 ppm for precursor ions and 1 Da for product ions. Enzyme specificity was limited to trypsin, with at least one K or R terminus required per peptide and up to four mis-cleavages allowed. Cysteine carboxamidomethylation, specified as a static modification, oxidation of methionine residues, was allowed, and phosphorylation was allowed on serine, threonine and tyrosine residues. Reverse decoy databases were included for all searches to estimate false positive rates, and data was filtered using a 5% false discovery rate in the Peptide Prophet module of SORCERER 2. Search results were further filtered by mass accuracy based on clustering of forward database assignments (−/+ 5 ppm). Results were also filtered using criteria specific to each PTMScan Direct Reagent, including whether a particular peptide was targeted by an antibody in the reagent or homologous to a target, a minimum MS1 intensity filter of 20,000 in at least one sample run, and a higher abundance of the peptide in the PTMScan Direct Reagent IAP than in a control IAP using empty Protein G beads.
Label-free quantification was performed using proprietary software as previously described [41,42,67]. MS1 peak intensities across all samples were retrieved from the ion chromatogram files using a mass precision of −/+ 5 ppm and a retention time window of 5 min. Retention time warping (or chromatographic alignment) was performed across binary comparisons to allow retrieval of the correct peak intensity. Peak intensities for all peptides that changed in abundance between treatments were manually reviewed in the ion chromatogram files to ensure accuracy and where necessary peak height measurements were replaced with peak areas. The pervanadate treated cell line mixture ("CST Cell Line" in Supplementary Tables S1–S4) was run as a separate sample with each study as a rich source of modified peptides. This allowed the label-free quantification software to search for all validated peptides across the experimental samples for a more complete quantitative analysis.
Protein-protein interactions from the STRING database (Version 9.0 [69]) were limited to the experimental, database and text-mining interaction categories with a minimum score of 0.700 ("high scoring" interactions in STRING). Interactions were imported into Cytoscape v2.8.1 for pathway map generation.
Protein concentrations were determined by Bradford assay using Coomassie Plus Protein Assay Reagent (Pierce), and total protein was normalized between samples. Samples were mixed with SDS-PAGE sample buffer (#7723, Cell Signaling Technology, Danvers, MA, USA) and run on 4%–20% gradient tris-glycine gels (Invitrogen). Proteins were transferred to nitrocellulose (Millipore) and blocked for 1 h in 5% nonfat dry milk (Sigma) in TBS. All primary and secondary antibodies were from Cell Signaling Technology and were used according to standard protocols available at www.cellsignal.com. Blots were developed on the Odyssey near-infrared imaging system (LI-COR). Gels were stained with GelCode Blue Stain Reagent (Thermo Scientific, USA #24592) and destained with deionized H2O.
The PTMScan Direct method used to evaluate signaling in response to UV damage of DNA in HeLa cells is broadly applicable across biological systems. In this study, the reagents allowed detection of endpoints indicative of activation of DNA damage checkpoint signaling and onset of apoptosis, both of which were confirmed by western blotting. Future work could include varying the UV dose or harvest time or using different cell lines. This could provide both a dose response and a temporal view of DNA damage signaling and induction of apoptosis in response to UV treatment.
PTMScan Direct Reagents can also be used to study other signaling areas, such as Akt/PI3K signaling, Ser/Thr and Tyr Kinase activation, or look broadly at critical signaling proteins across many different pathways using the PTMScan Direct: Multipathway Reagent [67]. These reagents are, therefore. powerful tools for the study of diseases, such as cancer or neurodegeneration, normal cellular processes, such as growth, development and differentiation, and the study of novel inhibitors and therapeutic agents to determine inhibitory profiles and potential off-target effects. The method combines the specificity of antibody-based methods with the ability to assay a large number of endpoints typical of LC-MS-based proteomic analysis.
Supplementary Materials (ZIP, 5751 KB)
The authors declare no conflict of interest.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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|Stephanie H.
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|Tuesday, July 3, 2012 5:09 PM
In addition to my regular Monday night group, for the summer, starting July 10th, I'll be running a group at the Boylston Library on Tuesday nights from 6:30 to 8:00pm.
There is no charge for the group and no need to sign up for a whole series...just drop in as you can!
Please let me know if you have any questions and/or are thinking of joining.
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**iwantfun rolled a random image posted in comment #18 at Photo bomb? ** this is what slender does when he catches you
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Hotels near Leicester Racecourse
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- Most bookedCollege Court
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"Clean hotel set in an old uni/college site. Room was small but clean. Breakfast outstanding with so much choice. Check-in process a little slow."
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"Suite was good, but surprised to have been charged extra for the sofa bed. Breakfast great with friendly and helpful staff"
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"Clean bedding, but the hotel lacks tea and coffee making facilities. Looking a bit tired but handy for the train station and town."
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